The Effect Of Dopamine On Gamma-aminobutyric Acid-activated Currents In Vitro Cultured Visual Cortical Neurons Of Rats

Chapter one Primary culture of visual cortical neurons of newborn ratsObjective To establish an experimental model of primary culture of visual cortical neurons of newborn rats and find a good way for the further investigation of ophthalmology diseases.Methods Primary culture of visual cortical neurons of newborn rats was performed by Dulbecco's modified Eagle's medium with 10% newborn calf serum and 10%F-12 Nutrient Mixtures and Neurobasal Medium with 2%B-27 Serum-Free Supplements.Evaluation of neurons was made by Nissl's staining.Results Cultured neurons grew well.They were satiety and with long apophysis.After Nissl's staining,the endochylema of neurons appeared amethyst.The structure of Nissl's body was clear.The endochylema of non-neuronal cells was scarcely pigmentosus.The purity of neurons was 40%～50%.Conclusions 1.Cultured neurons grew well.They were satiety and with long apophysis.2.The purity of neurons was 40%～50%. Chapter two Electrophysiological characteristics of GABA-activated currents in vitro cultured visual cortical neurons of ratsObjective To study the electrophysiological characteristics of GABA-activated currents in vitro cultured visual cortical neurons of rats.Methods The patch clamp was used to record the whole cell currents of GABA-A channels in vitro cultured visual cortical neurons of 11-14d of the neonatal rats.Results 1.It was found that majority(85.42%,41/48)of the examined neurons were sensitive to GABA.GABA could induce an inward current which was dose dependent.2.GABA-activated currents had an obvious desensitization.The recovery time of GABA-A receptors from desensitization was 5 minutes.3.GABA-activated currents could be blocked by bicuculline and simulated by muscimol.4.The reversal potential of GABA-activated currents was about 0mV which was near the equilibrium potential of Cl~-.5.The width of GABA-activated currents was stable.The average variance was 4.51±3.02%. Conclusions 1.Majority of the cultured neurons were sensitive to GABA.2.GABA-activated currents were dose dependent and had an obvious desensitization.3.The width of GABA-activated currents was stable. The average variance was 4.51±3.02%.Chapter three The effect of dopamine on GABA-activated currents in vitro cultured visual cortical neurons of ratsObjective To study the effect of dopamine on GABA-activated currents in vitro cultured visual cortical neurons of rats.Methods The patch clamp was used to record the whole cell currents of GABA-A channels in vitro cultured visual cortical neurons of 11-14d of the neonatal rats.When neurons were treated respectively with different density of dopamine,SKF38393,quinpirole,and the combination of SKF38393 and SCH23390,SKF38393 and Quinpirole prior to the application of GABA for some time,GABA-activated currents were observed.Results 1.When dopamine was added,GABA-activated currents were inhibited and showed a dose and time dependent change.When the density of dopamine was 10μM～500μM,the changes of GABA-activated currents had significant difference with contral group(P＜0.05),whereas 50μM dopamine inhibited most markedly.After the application of dopamine for 2～6min,the changes of GABA-activated currents had significant difference with contral group(P＜0.05).The inhibitory action was stable after the application of dopamine for 4min.2.When SKF38393 was added,GABA-activated currents were inhibited and showed a dose and time dependent change.When the density of SKF38393 was 1μM～100μM,the changes of GABA-activated currents had significant difference with contral group(P＜0.05).The inhibitory action was stable when the density of SKF38393 exceeded 50μM.After the application of SKF38393 for 3～6min,the changes of GABA-activated currents had significant difference with contral group (P＜0.05).The inhibitory action was stable after the application of SKF38393 for 4min.SCH23390(10μM)could significantly depress the effect of SKF38393(P＜0.05).3.After application of quinpirole(10μM,100μM,1mM) for 5min and 10min respectively,GABA-activated currents had no changes.4.Compared with using SKF38393 alone,combination of SKF38393(10μM)and quinpirole(10μM)for 4～6min could weaken the inhibition of GABA-activated currents,whereas the difference was significant after application for 5min(P＜0.05). Conclusions 1.When dopamine or SKF38393 was added, GABA-activated currents were inhibited and showed a dose and time dependent change.SCH23390 could significantly depress the effect of SKF38393.2.Quinpirole had no effect on GABA-activated currents.3.Combination of SKF38393 and quinpirole could weaken the inhibition of GABA-activated currents.